1. Field of the invention
This invention relates to a method of selecting a strain of virus use as a component of a vaccine against influenza, to a method of preparing such a vaccine and to the vaccine when prepared by the invented method.
2. Description of prior art
Influenza is still a major disease in man, primarily because of the property of antigenic variation associated with influenza virus. There are three types of influenza viruses (A, B, and C) that are defined by the absence of serologic crossreactivity between their internal proteins. Influenza A viruses are further classified into sub-types based on antigenic differences of their glycoproteins, the haemagglutinin (HA or H) and neuraminidase (NA or N). The most important types of influenza virus in humans currently are type A sub-types H1N1 and H3N2 and type B. These are therefore the types which are incorporated in influenza vaccines.
The haemagglutinin (HA) antigens of influenza viruses A and B undergo frequent and progressive changes in antigenic specificity, whereby the virus seeks to evade neutralisation by the immune response of the human body. This "antigenic drift" results from changes in the amino acid sequence of HA and NA antigens, brought about by natural selection, presumably under pressure of the immune response. It is well known that vaccination with a particular strain of influenza virus, which is isolated from a particular outbreak of influenza, is unlikely to confer complete protection against a future outbreak. New vaccines are, therefore, continually being prepared from recent isolates to counter the latest antigenic drift. With influenza A viruses, this incorporation of new strains in the vaccine is at most a yearly occurrence.
It is the practice in preparing influenza vaccines on a commercial scale to grow an isolated strain in embryonated hens' eggs. Initially the virus is recovered from a throat swab or similar source and isolated in eggs. The initial isolation in eggs is difficult, but the virus adapts to its egg host and subsequent propagation in eggs takes place relatively easily. In contrast, while the initial isolate can usually be made in mammalian tissue culture, using, for example, the well known canine kidney cells known as MDCK, mammalian tissue culture has not been favoured for the large scale production of influenza vaccines, because of fears about possible adverse effects of the use of transformed cells. Such cells are currently not licensed for use as a substrate in vaccine production. Furthermore, there are production problems in the use of alternative cells such as human diploid cells.
Because of antigenic drift of the most important single influenza virus protein, the haemagglutinin (HA), the suitability of current virus strains for vaccines must be continually reassessed. This has been performed for the last 3 decades by analysing the antigenic structure of the HA of field strains isolated and cultivated in embryonated hens' eggs. Usually a number of such candidate virus strains are screened versus polyclonal ferret and human antisera and more recently monoclonal antibodies using the haemagglutination inhibition (HI) test. A virus which reacts poorly and to low frequency with human sera is commonly selected and such a virus (or a reassortant containing the HA and NA genes of the virus) is further passaged in eggs to produce virus in quantities for inactivated vaccine. It has been thought that the criterion of poor reactivity is indicative of a virus which has undergone antigenic drift, and against which the human population would show little resistance. Such a virus could cause a future epidemic and so might be the best choice for use in vaccines.
Doubts about the efficacy of some influenza vaccines have long been expressed by clinicians. These have been given added weight by recent evidence derived from antigenic comparison of egg-adapted virus with virus grown exclusively in mammalian tissue culture. Using both monoclonal and polyclonal antibodies in haemagglutination-inhibition (HI) and virus neutralisation assays. G. C. Schild et al., Nature 303, 706-709 (1983) and in "The Molecular Virology and Epidemiology of Influenza" (Ed. Sir Charles Stuart-Harris and Professor C. W. Potter), Academic Press, London, New York and Orlando, (1984), pages 163-174 have demonstrated that egg-adapted virus is antigenically distinct from virus from the same clinical sample grown solely in tissue culture (MDCK) cells. In addition, MDCK-derived influenza B or A (H1N1) virus detects antibody more frequently and to a higher titre in post-infection human sera using haemagglutination-inhibition and neutralisation tests than does egg-adapted virus. G. C. Schild and his colleagues J. S. Oxford and J. S. Robertson at the National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertford, EN6 3QG, have described these findings as having serious implications in relation to the continued use of egg-adapted viral strains for epidemiological studies, virus research and possibly vaccine production.
The haemagglutinin (HA) antigen provides the major antigenic determinants of influenza virus. Its role is to attach the virus to the host cell and thereafter intracellularly to fuse the envelope of the virus to the cell membrane. The three-dimensional structure of an H3 sub-type of HA is established. Antigenic sites for HA of H1N1 and H3N2 and of influenza B have been identified and are located predominantly on the surface of the "globular head" of the protein. The site at which the HA attaches to the cell has also been identified as on the globular head.
The nucleotide and amino acid sequences of many HAs are known. Thus G. Winter et al., Nature 292, 72-75 (1981) have sequenced HA from the H1 sub-type, and compared it with that of the H2 and H3 sub-types of influenza A, while M. Krystal et al., Proc. Natl. Acad. Sci. USA 79, 4800-4804 (1982) have sequenced HA of a type B virus, and F. L. Raymond et al., Virology 148, 275-287 (1986) have analysed the HA nucleotide sequence in the globular head region for 19 strains of type A sub-type H1 isolated from 1950 to 1983. It is customary, in this field, to use an amino acid numbering system based on an H3 sub-type as set forth by G. Winter et al., supra., at page 74. The numbering system allows for deletion and insertion differences in H1, H2 and H3 sub-types of serotype A and is also used for type B. This numbering system is used in this specification, except where otherwise stated.
J. S. Robertson et al., have given several papers in 1984 and 1985 analysing the amino acid difference in HA between strains of virus (a) isolated and grown in cells and (b) isolated in cells and subsequently egg-adapted. The paper given orally at Cambridge, England in September 1985 and entitled "Characterisation of egg-adapted variants of human influenza viruses" is believed representative. This paper has been published in "The Biology of Negative Strand Viruses" (Ed. B. W. J. Mahy and D. Kolakofsky), Elsevier Biomedical Press (1987), pages 412-416. It is reported that a feature common to the HA of all egg-adapted viruses examined is the location of amino acid substitution(s) adjacent to the receptor binding site. In A(H1N1), residues 138, 187, 189, 190 and 225 are all located in the proximity of the receptor binding site. Residue 163 which forms part of a glycosylation site in the HA of cell culture derived H1N1 virus, is located on the opposite side of the molecule from the receptor binding site; however the presence of a carbohydrate side chain on the N (asparagine) residue at 163 might affect the receptor specificity of an adjacent HA molecule. In A(H1N1) some of these changes (residues 163, 190 and in some cases 225) did not appear to alter the antigenicity of the HA whilst others did (residues 187, 189 and in some cases 225), and some (residues 65, 138, 300) occasionally accompanied substitution at 187 and 189 and may have contributed towards the altered antigenicity of these variants. In the B serotype, see also J. S. Robertson et al., Virology 143, 166-174 (1985),. HA residues 196-198 in B type nomenclature (equivalent to 187-189 in H3 numbering) constitute a glycosylation site. Egg adaptation of B type virus is accompanied by an amino acid substitution of either N at 196 or T at 198 and a concomitant change in antigenic profile.
In an oral paper given by Dr. J. S. Robertson at the Department of Cellular, Viral and Molecular Biology, University of Utah, Salt Lake City, USA, 26th Apr., 1985 and at a symposium at Keystone, Colorado, USA organised by the University of California, Los Angeles, USA given in April 1985, the antigenic profiles of virus grown in MDCK cells and then egg-adapted have been compared with an MDCK cell-grown virus in a HI test using a test set of several different monoclonal antibodies and human and ferret polyclonal antisera. Some of the egg-adapted viruses could not be distinguished antigenically from cell-grown virus and were termed "cell-like". Others were found to have a different profile which could not be distinguished from that obtained when the initial isolate was made in eggs. These were termed "egg-like". Yet a third group differed from the others and was generally not very reactive towards any of the antibodies. These were termed "odd". These antigenic changes occurring upon passage of the MDCK cell-isolated virus in eggs were correlated with HA amino acid substitutions. In none of these various prior papers have any conclusions been reached concerning what should be done to make influenza vaccines more effective.
J. L. Lathey et al., Journal of Medical Virology 19, 155-159 (1986) have shown that antigenic extract from cell-grown virus, grown in African green monkey kidney cells, is superior to that from egg-grown virus for diagnosis of the course of an infection of influenza B. These authors noted a change in antigenicity when an egg-grown virus was subsequently passaged in the monkey kidney cells, and put forward the idea that the change in amino acid sequence induced by egg passage might be reversible upon subsequent passage in mammalian cells.
S. Patterson and J. S. Oxford recently reviewed the interactions between viruses and host cells in a paper in Vaccine 4, 79-90 (1986). Table 1 of this paper shows the result of an HI test of three monoclonal antibodies to assess the antigenic differences between viruses having various histories of isolation and passage. After discussing these antigenic differences, they comment on the relevance of their results to vaccines. Noting that the antisera of convalescent influenza sufferers react better with viruses cultivated in cells rather than in eggs, they state (page 81, right-hand column) that they are investigating the potential of influenza vaccines prepared in mammalian cells.
The above-mentioned prior disclosures of Robertson et al. were regarded by the present inventors as of scientific interest, in showing that a virus isolated in animal cells and passaged in eggs might remain "cell-like" in antigenic character. This complemented the earlier finding of Schild et al. in the above-mentioned 1983 paper in Nature that a virus isolated in eggs and passaged in cells remained "egg-like" in antigenic character. It seemed to the inventors that growing viruses in eggs for the production of vaccine strains, according to the conventional method, might alter the antigenic profile characteristic of the virus present in a specimen taken from a patient suffering from influenza. Therefore, it seemed to the inventors, it might be better to isolate and grow vaccine strains in animal cells. Given the worries about the use of transformed cells (see above), one would have to use human diploid cells or some other non-transformed animal cells. Production difficulties would be anticipated with such cells and viruses would only be produced at low titres.